EP0664002A1 - Procede permettant de determiner des necroses du muscle cardiaque a l'aide d'anticorps diriges contre le peptide n-terminal de la troponine i - Google Patents

Procede permettant de determiner des necroses du muscle cardiaque a l'aide d'anticorps diriges contre le peptide n-terminal de la troponine i

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Publication number
EP0664002A1
EP0664002A1 EP94903863A EP94903863A EP0664002A1 EP 0664002 A1 EP0664002 A1 EP 0664002A1 EP 94903863 A EP94903863 A EP 94903863A EP 94903863 A EP94903863 A EP 94903863A EP 0664002 A1 EP0664002 A1 EP 0664002A1
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EP
European Patent Office
Prior art keywords
peptide
antibody
seq
amino acid
amino acids
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP94903863A
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German (de)
English (en)
Inventor
Helmut Lill
Frédéric DONI
Anneliese Borgya
Christoph Seidel
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Roche Diagnostics GmbH
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Roche Diagnostics GmbH
Boehringer Mannheim GmbH
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Publication of EP0664002A1 publication Critical patent/EP0664002A1/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4716—Muscle proteins, e.g. myosin, actin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans

Definitions

  • the invention relates to a method for the determination of cardiac muscle necrosis by means of antibodies against the N-termina troponin I peptide, in which a blood sample with an antibody against the N-terminal troponin I peptide is added and the complex which is formed is detected and the complex formed therein drive antibodies used and a method for the production of such antibodies.
  • the myofibrils of the striated muscle consist of two interacting protein filaments, the thick filament with myosin as the main component and the thin filament containing actin, tropomyosin and troponine.
  • Troponin is a complex structural component in the cells and consists of three different proteins:
  • Troponin C binds calcium ions
  • Troponin I an actin binding subunit
  • Troponin T attaches to tropomyosin.
  • troponin Three isoforms of troponin I are known, corresponding to their occurrence in the heart muscle (Tnlc), slow skeletal muscles (Tnls) and fast skeletal muscles (Tnlf). Cardiac troponin I differs from skeletal troponin I mainly by an additional N-terminal sequence of approx. 30 amino acids.
  • Cardiac troponin I is released after an acute myocardial infarction and can be detected in the blood plasma. It thus represents a marker for myocardial cell necrosis. Cummins, J. Mol. Cell. Card. 19 (1987), 999-1010 and Cummi and Cummins, Clin. Invest. 113 (1987), 1333-1344 could prove that Tnlc in serum normally occurs in concentrations of 10 ng / ml. It was shown that an increased serum concentration z can be observed in transmural infarction. Tnlc was increased from the 4th to 168th hour after the onset of pain in 37 patients with acute transmural infarction. Similar results were obtained in the animal model. Thereafter, the time interval for the absolute diagnostic sensitivity for Tnlc is the 10th - 50th hour after the infarction event.
  • MAK monoclonal antibodies
  • Tnlc monoclonal antibodies
  • some of the MAK bind the MAK 3C6 or 11E12 in the N-terminal region of Tnlc. None of these antibodies recognize an epitope between amino acids 14 to 24 well.
  • the MAK 11E1 obviously recognizes an epitope between the amino acids 14 to 37.
  • this MAK requires the amino acids N and Y in position 25 and 26, that is to say its epitope lies approximately between the amino acids 21 to 30. The same applies to the others published MAKs. Bodor et al., Clin. Chem.
  • Tnlc is subject to proteolytic cleavage, which can take place both intra- and extracellularly.
  • an N-terminal fragment of Tnlc can be cleaved off here.
  • Tnl recovery may be too low in the tests described so far, since the epitopes are separated from antibodies between which there is a cleavage site.
  • the object of the present invention was to develop a determination method which avoids false negative results in the Tnlc determination.
  • Another object of the invention is an antibody which recognizes the N-terminal peptide of cardiac troponin I and is obtainable by immunization with an immunogen which contains a peptide of the amino acid sequence SEQ ID NO 1 or at least a partial sequence thereof with at least 6 amino acids in length and Isolation of the antibody from the serum of the immunized animals by known methods and a monoclonal antibody which recognizes the N-terminal peptide of cardiac troponin I and is obtainable by immunization with an immunogen which is a peptide of the amino acid sequence SEQ ID NO 1 or at least one Contains partial sequence thereof with at least 6 amino acids in length, immortalization of the spleen cells of the immunized animals, cloning of those immortalized cells that produce the desired antibody and isolation of the antibody from d cloned cells by known methods.
  • Another object of the invention is the use of synthetic peptides of the amino acid sequence SEQ ID NO 1 or at least a partial sequence thereof with at least 6 amino acids in length as an immunogen for the production of polyclonal and monoclonal antibodies, as standard material, polyhapten or labeled antigen in immunoassays.
  • a synthetic peptide is to be understood as meaning peptides which have been produced by chemical synthesis as well as by recombinant production.
  • the immunological determination method for cardiac muscle necrosis is carried out using methods familiar to the person skilled in the art.
  • the determination in samples, ie body fluids such as blood, plasma or serum can be carried out by a radio immunoassay, enzyme immunoassay chemiluminescence immunoassay or by immunofluorescence. All known methods such as competitive immunoassays, sandwich immunoassays and IE methods or homogeneous immunoassays such as CEDIA ® can be used as a method variant. It is possible to carry out the test in the form of a wet test or dry test. In some cases it is advantageous to denature the sample before or during the determination.
  • the Tnlc-specific body is immobilized on a solid phase.
  • the immobilization can be done adsorptively, covalently or via a specific binding such as biotin / streptavidin, antibody / antigen or sugar / lectin.
  • the Tnlc-specific antibody according to the invention is bound to one of these binding partners.
  • the other binding partner is bound to the solid phase.
  • materials such as. B. tubes, plastic cuvettes, microtiter plates, spheres, magnetic particles or microcarries made of plastics such as polystyrene, vinyl polymers, Polypropylene, polycarbonate, polysaccharides, silicones, rubber or treated glass (see, for example, ET Maggio, Enzyme Immunoassays, CAC. Press, Florida (1980), in particular pages 175-178, EP-A-0 063 064, Bioengineering 16 (1974) , 997-1003, CJ Senderson and DV Wilson, Immunology 20 (1971), 1061-1065).
  • plastics such as polystyrene, vinyl polymers, Polypropylene, polycarbonate, polysaccharides, silicones, rubber or treated glass
  • a carrier material coated with avidin or streptavidin, in particular polystyrene, preferably produced as described in EA-0 269 092, is used as the solid phase.
  • the usual nonwovens such as cellulose or glass fiber nonwovens as well as membranes are to be used as the solid phase in a dry test.
  • the sandwich test has proven particularly useful. At least one antibody according to the invention must be used.
  • the second antibody can be a further antibody according to the invention, but an antibody of the prior art can also be used. The prerequisite is that the two antibodies do not, or only insignificantly, cross-react with one another.
  • An immobilized antibody which recognizes the N-terminal peptide of cardiac troponin I is preferably used in combination with a second Tnlc-specific antibody to which a determinable group is coupled. Common label groups such as enzymes or fluorescent or luminescent residues can be used as a definable group.
  • the second antibody which also preferably recognizes the N-terminal peptide of Tnlc, is selected so that it does not or only insignificantly interferes with the binding of the first antibody to this peptide.
  • Both antibodies can be directed against different epitopes on the N-terminal peptide of Tnlc.
  • the test can be carried out successively, ie an antibody is first incubated with the sample before the second antibody is added, or simultaneously, ie all the reactants are added simultaneously in the test. Since the N-terminal peptide of Tnlc can be phosphorylated on the two serine residues in positions 23 and 24, an anti-phosphorus serine antibody can be used as a second antibody in the sandwich immunoassay. Anti-phosphorus-serine antibodies are known per se.
  • Another preferred embodiment is the competitive test.
  • the use of a synthetic peptide as the labeled antigen in the test is preferred because it always has the same and therefore standardizable composition and, in contrast to the naturally occurring Tnlc, is not unstable.
  • TINIA the turbidometric metric inhibition immunoassay
  • a sample is mixed with a polyhapten, which consists of a carrier, for example a late particle or dextran and attached N-terminal Tnl peptides of the amino acid sequence SEQ ID NO 1 or at least a partial sequence thereof with a length of at least 6 amino acids, exists and incubated the antibodies of the invention.
  • Immune complexes are formed from polyhapten or Tnlc and antibodies. The binding of the antibodies to the polyhapten leads to aggregates that can be measured by increasing the turbidity.
  • the binding of antibodies to the free analyte does not lead to any major aggregates, which therefore cause turbidity, since the epitopes recognized by the antibodies according to the invention occur only once on the analyte.
  • the agglutination of the polyhapten is thus partially inhibited.
  • the exact content of analyte in a sample can be determined using a standard curve.
  • the antigen which contains a peptide of the amino acid sequence SEQ ID NO 1 or at least a partial sequence thereof with at least 6 amino acids in length is coupled to ED.
  • An antibody in the sense of the invention is to be understood as a complete antibody or bi- or monovalent fragments thereof.
  • the antibodies can be both monoclonal and polyclonal. The use of monoclonal antibodies is preferred.
  • Another object of the invention is a process for the production of polyclonal antibodies which recognize the N-terminal peptide of cardiac troponin I and the antibodies obtainable therewith.
  • the method consists in that ma test animals, preferably sheep, with an immunogen which contains the amino acid sequence according to SEQ ID NO 1 or a partial sequence thereof with at least 6 amino acids in length Combination with an adjuvant, immunized at least four times at four- to six-week intervals over several months (preferably 4-6 months), crude serum is obtained and this is purified using methods known to the person skilled in the art.
  • Adsorbers are used for immunoadsorption which have a peptide m of the amino acid sequence according to SEQ ID NO 1 or a partial sequence thereof with at least 6 amino acids in length covalently bound.
  • Another object of the invention is a process for the production of monoclonal antibodies which recognize the N-terminal peptide of cardiac troponin I and the antibodies obtainable therewith.
  • the method consists in m experimental animals, preferably inbred mice, with an immunogen which contains the amino acid sequence according to SEQ ID NO 1 or a partial sequence thereof with at least 6 amino acids in length, in combination with an adjuvant, over several months with at least four immunizations immunized intraperitoneally at four- to six-week intervals, spleen cells obtained and fused with a permanent myeloma cell line, the clones isolated and the antibodies obtained therefrom.
  • Aluminum hydroxide is preferably used as an adjuvant together with Bordetella pertussis or Freund's adjuvant.
  • the immunogen which contains the amino acid sequence according to SEQ ID NO 1 or a partial sequence thereof with a length of at least 6 amino acids, is preferably coupled to a carrier protein to increase the antigenicity.
  • Several different partial sequences can also be used in combination in an immunization.
  • hybrid cells obtained in the fusion according to the known method of Köhler and Milstein are obtained in the usual way, for example using a commercially available cell sorter or by "limiting dilution "cloned.
  • Cultures that react positively to the N-terminal peptide of cardiac troponin I are processed in each case.
  • hybridoma cell lines are obtained which produce the monoclonal antibodies according to the invention. These cell lines can be cultivated according to known methods and the monoclonal antibodies produced by them can be isolated.
  • the polyclonal or monoclonal antibodies according to the invention are used in all known immunochemical processes, for example in immunoassays or immunohistology. They are ideally suited for the specific determination of cardiac muscle necrosis in a sample, for example serum or plasma. False negative results can be minimized or avoided in this way.
  • the antibodies as such or fragments thereof which have the appropriate immunological properties, for example F (ab) 2 or Fab fragments, can be used for these determination methods. The term antibody is therefore understood to mean both complete antibodies and their fragments.
  • an antigen which is a pep of the amino acid sequence is used as the immunogen, screening reagent in the selection of the monoclonal antibodies, standard material in the immunoassay, polyhapten in the TINIA test or as the labeled antigen in an immunoassay
  • the antigens ie the amino acid sequences, have proven to be particularly suitable
  • SEQ ID NO 3 SDAAREPRPA
  • the amino acid sequence SEQ ID NO 1 corresponds to the 30 N-terminal amino acids of human cardial Tnl.
  • the sequence v human Tnlc is known from Vallins et al., FEBS Letters 270 (199 57-61. It was surprising, however, that by using antibodies which were produced by means of peptide immunogens, the peptides of the amino acid sequence SEQ NO 1 or at least one Partial sequence of which contain at least amino acids in length, cardiac muscle necrosis could be detected, samples which were assessed negatively in the determination of total Tnlc, since the measurement signal was just below the "cut-off", ie the measurement signal which was just as positive was evaluated, were still recognized as positive.
  • Another object of the invention is a test kit for determining cardiac muscle necrosis, which contains at least one polyclonal or monoclonal antibody according to the invention in a container.
  • the other components and reagents necessary for carrying out the method according to the invention such as the solid phase, which can be coated with streptavidin, are further antibodies according to the invention which can be labeled, polyhaptens, auxiliary substances such as buffers or Anti-interference reagents included.
  • the invention furthermore relates to a test kit which contains a standard for use in the determination of Tnlc, which contains a peptide of the amino acid sequence SEQ ID NO 1 or at least a partial sequence thereof.
  • example 1 The invention is illustrated by the following examples: example 1
  • the peptide was synthesized on 180 mg of 4- (2 ', 4'-dimethoxyphenyl-Fmoc-aminomethyl) -phenoxy resin with a loading of 0.45 mmol.
  • the sequence was constructed with a combination of Fmoc as temporary and P c / tBu / Trt as permanent protective groups. 16 equivalents of the following amino acid derivatives were used:
  • the coupling reactions were carried out with 1.1 equivalents of di-propylcarbodiimide / N-hydroxybenzotriazole in relation to the amino acid derivatives as activation reagents.
  • the coupling times were 90 minutes and were carried out twice with 8 equivalent amino acid derivatives each.
  • the temporary protective group was removed by means of piperidine within 20 minutes.
  • the reactions were carried out in dimethylformamide, and the same solvent was used for the washing steps that followed the reaction steps.
  • the synthesis was carried out fully automatically on a Zinsser SMPS 350 in 6 tubes of 30 mg synthesis each carried out.
  • the peptide was released with the elimination of the side chain protective groups, except Acm, with a mixture of 95% trifluoroacetic acid, 3% ethanedithiol and 2 anisole within 90 minutes.
  • the cleavage solution was filtered off with 10 times the amount of diisopropyl ether. added and the precipitate formed is filtered off.
  • the precipitate was washed with diisopropyl ether, dissolved in 20% acetic acid and lyophilized. 107 mg of white lyophilisate were obtained.
  • the purity was checked by means of HPLC (92% area), the identity was checked by means of mass spectroscopy (MH +: 1603).
  • Fmoc- fluorenylmethyloxycarbonly- -tBU: tertiary butyl- Trt-: trityl- Acm-: acetamidomethyl-
  • peptide immunogens comprising amino acids 1-10, 6-15, 11-20, 16-26 and 1-30 of the N-terminal troponin I was prepared using the following peptides:
  • KLH Keyhole limpet hemocyanin
  • the peptide is derivatized with 6-maleimidohexanoic acid, Ac -protected and MH-derivatized peptide can then be distinguished on HPLC based on different retention times (Vydac C18, 5 ⁇ m, 300 A, 4.6 x 250 mm; 0-45% B in 30 minutes; A: 0.1% TFA in H 2 0; B: 0.1% TFA in acetonitrile / H 2 65:35).
  • reaction mixture is loaded onto a filtration column (AcA 202, 24 x 4 cm, IBF Biotechnology) and the protein / peptide conjugate is eluted with the above-mentioned phosphate buffer.
  • the protein concentration is again determined by BCA, the peptide coupling efficiency using Ellman's test. 14.8 mg (72% of theory) of the immunogen were obtained with a loading of 547 peptide / KLH-MH.
  • the peptide was synthesized on 30 mg of 4- (2 ', 4'-dimethoxyphenyl-Fmocaminomethyl) phenoxy resin with a loading of 0.45 mmol /.
  • the sequence was constructed with a combination of Fmoc as temporary and Pmc / tBU / Trt / DmTr / Boc as permanent protective groups. 16 equivalents of the following Fmoc amino acid derivatives were used:
  • the coupling reactions were carried out with 1.1 equivalents of diisopropylcarbodiimide / N-hydrobenzotriazole in relation to the amino acid derivatives as activation reagents.
  • the coupling times were 90 minutes and were carried out twice with 8 equivalents of amino acid derivative.
  • the temporary protective group was removed using piperidine within 20 minutes.
  • the reactions were carried out in dimethylformamide, and the same solvent was used for the washing steps which followed the reaction steps iy
  • the synthesis was carried out fully automatically on a Zinsser SMPS 350.
  • the peptide was released with elimination of the side chain protective groups with a mixture of 95% trifluoroacetic acid, 3% ethanedithio and 2% anisole within 90 minutes.
  • the cleavage solution was filtered off, 10 times the amount of diisopropyl ether was added and the precipitate was filtered off.
  • the precipitate was washed with diisopropyl ether, dissolved in 20% acetic acid and lyophilized. 22.5 mg of white lyophilizate were obtained.
  • the purity was checked by HPLC (% Area), the identity was checked by mass spectroscopy (MH +: 1996).
  • Balb / c mice 8-12 weeks old, were immunized intraperitoneally with 100 ⁇ g Pepti immunogens from Example 2, with a complete Freund's adjuvant. After 6 weeks, three further immunizations were carried out at 4-week intervals. In each case 50 ⁇ g immunogen, adsorbed on aluminum hydroxide and Bordetella pertussis, were administered intraperitone. One week after the last immunization, blood was drawn and the antibody titer in the serum of the test animals was determined. If the course of the immunization was positive, fusion was carried out. Four days before the fusion, one more intravenous immunization was carried out with 50 ⁇ g immunogen in phosphate-buffered saline.
  • the primary cultures which contained antigen-specific antibodies, go into the single cell depot with the aid of a fluorescence-activating cell sorter (FACS).
  • FACS fluorescence-activating cell sorter
  • the clones obtained were cultivated and tested for specificity in the ELISA method.
  • Hybridoma cell lines were established from positive clones and larger amounts of antibodies were produced using cell fermenters or ascites production. -
  • Microtiter plates (Nunc company) were coated with streptavidin and loaded with 1 ⁇ g / ml biotinylated peptide. After a washing step, the antibody sample was incubated. After another washing step, the detection antibody was incubated with polyclonal sheep anti-mouse Fcg, Fab peroxidase conjugate. After a further washing step, the peroxidase activity was determined using a color substrate (ABTS).
  • ABTS color substrate
  • the specificity of the binding of the antibody sample to the biotinylated peptides was checked by inhibition with free peptide.
  • the antibody sample was incubated with 10 ⁇ g / ml peptide with the amino acid sequence SEQ ID NO 1 before the antibodies were used in the above microtiter plate test. A greatly reduced binding under these conditions was only obtained from specific antibodies.
  • the cell clone MAK ⁇ TnIc peptide, II-20> M-7.3.1 was obtained by immunizing mice with the peptide SEQ ID NO 4 coupled to KLH and by cloning according to Example 4.
  • the monoclonal antibodies produced by these cells are distinguished by the fact that they recognize very specifically an epitope of the N-terminal peptide of Tnlc which can be seen from Table 1. Analysis of the peptides bound by the antibody reveals that this antibody recognizes, as a core epitope, a linear amino acid sequence which corresponds to positions 11 to 19 of SEQ ID NO 1.
  • the cell clone MAK ⁇ TnIc peptide, 6-15> M-3.10.3 was obtained by immunizing mice with the peptide SEQ ID NO 3 coupled to KLH and by cloning according to Example 4.
  • the monoclonal antibodies produced by these cells are distinguished by the fact that they recognize very specifically an epitope of the N-terminal peptide of Tnlc which can be seen from Table 1.
  • Analysis of the peptides bound by the antibody reveals that this antibody recognizes, as a core epitope, a linear amino acid sequence which corresponds to positions 7 to 12 of SEQ ID NO 1.
  • this antibody is particularly suitable for the detection of cardiac muscle necrosis. It can be used in both competitive assays and sandwich immunoassays.
  • the peptides were 12-amino acid partial sequences of the N-terminal sequence of cardiac troponin I (SEQ ID NO 1) and each differed by one amino acid so that the entire sequence could be covered.
  • Streptavidin-coated microtiter plates (microcoat) were loaded with 100 ⁇ l / well of the following biotinylated peptides (1 ⁇ g / ml each): 5-16, 6-17, 7-18, 8-19, 9-20, 10-21, 11 -22, 12-23, 13-24, 14-25 and 15-26, prepared analogously to Example 3 (the numbers given relate to SEQ ID NO 1 and denote the first and last position of the partial amino acid sequence of the respective peptide ).
  • the absorption is measured after 30-60 min at 405 nm.
  • MAK development with Immunogen SEQ ID NO 2 was discontinued because suitable PAKs are available.
  • the peptide was synthesized on 120 mg of 4- (2 ', 4'-dimethoxyphenyl-Fmocaminomethyl) phenoxy resin with a loading of 0.45 mmol / g.
  • the sequence was constructed with a combination of Fmoc as temporary and Pmc / tBU / Trt / OtBu as permanent protective groups. 16 equivalents of the following Fmoc amino acid derivatives were used:
  • the coupling reactions were carried out with 1.1 equivalents of diisopropylcarbodiimide / N-hydrobenzotriazole in relation to the amino acid derivatives as activation reagents.
  • the coupling time was 90 minutes and was carried out twice with 8 equivalents of amino acid derivative.
  • the temporary protective group was removed using piperidine within 30 minutes cleaved.
  • the reactions were carried out in diethylformamide, and the same solvent was used for the washing steps which followed the reaction steps.
  • the synthesis was carried out fully automatically on a Zinsser SMPS 350.
  • the peptide was released with elimination of the side chain protecting groups with a mixture of 95% trifluoroacetic acid, 3% ethanedithiol and 2% anisole within 90 minutes.
  • the cleavage solution was filtered off, 10 times the amount of diisopropyl ether was added and the precipitate was filtered off. The precipitate was washed with diisopropyl ether, dissolved in 20% acetic acid and lyophilized. 29.5 mg of white lyophilizate were obtained. The purity was checked by means of HPLC (% area).
  • the crude peptide was passed over a 20 mx 300 ⁇ m RP-HPLC column (column material: C-18, 5 ⁇ , 300 A) with a gradient system from 0% B to 100% B in 100 minutes (Puff A: 0.1% trifluoroacetic acid in water, Buffer B: 0.1% trifluoroacetic acid in water / acetonitrile 40:60) to a purity according to HPLC of 97%.
  • Microtiter plates (Nunc-Maxisorb) were coated overnight at 4 ° C. with 100 ⁇ l 5 ⁇ g / ml PAK ⁇ Dig> S (IS) (Boehringer Mannheim CAT.-NO. 1330713), with 200 ⁇ l / well PBS / 1% RSA reload and washed 3 times.
  • Table 3 shows the results obtained with sera from normal people (NS) and people after myocardial infarction (MI). A clear distinction could be made between the two groups.
  • the serum of a patient with myocardial infarction marked with * showed a signal which deviated significantly from the normal sera only in one test according to the present patent. In other test versions, which recognize Tnlc and not the N-terminal peptide, this serum could hardly be distinguished from the normal sera.
  • Tnlc solution (0; 0.4; 2; 5; 10; 20; 40; 100 ng / ml human cardial troponin I, double concentrated) or sample (sera from normal people (NS) or from people ( MI) after a heart attack, diluted 1 + 1) pipetted.
  • 60 ⁇ l / tube of wall antibodies (5 ⁇ g / ml MAK ⁇ TnIc peptide, 6-15> M- 3.10.3-IgG-Bi, 4-fold concentrated) are added.
  • the monoclonal antibody was obtained by purification according to the prior art with ProteinA-Sepharose and derivatized with Biotinoyl- ⁇ -aminocaproic acid-N-hydroxysuccinimide.
  • the absorption is measured after 30-60 min at 405 nm.
  • the results of the sandwich immunoassay with Tnic solution as analyte are shown in Table 4 and Figure 2/2. The measurement signal increases depending on the amount of analyte.
  • Table 5 shows the results obtained with sera from normal people (NS) and people after myocardial infarction (MI). A clear distinction could be made between the two groups.
  • the serum of a patient with a myocardial infarction marked with * showed a signal that differed significantly from the normal sera only in one test according to the present patent. In other test versions, which recognize Tnlc and not the N-terminal peptide, this serum could hardly be distinguished from the normal sera.

Abstract

Selon un procédé de détermination immunologique de nécroses du muscle cardiaque, on mélange un échantillon de sang avec un anticorps qui reconnaît le peptide N-terminal de la troponine I cardiaque et que l'on obtient par immunisation à l'aide d'un immunogène qui contient la séquence d'aminoacide SEQ ID 1 ou certaines de ses séquence partielles ayant une longueur d'au moins 6 aminoacides, on isole l'anticorps polyclonal ou monoclonal à partir du sérum ou à partir de cellules de rate immortalisées et clonées des animaux immunisés, selon des méthodes connues, et le complexe qui se forme est détecté de manière appropriée.
EP94903863A 1992-12-23 1993-12-22 Procede permettant de determiner des necroses du muscle cardiaque a l'aide d'anticorps diriges contre le peptide n-terminal de la troponine i Withdrawn EP0664002A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE19924243648 DE4243648A1 (de) 1992-12-23 1992-12-23 Verfahren zur Bestimmung von Herzmuskelnekrosen mittels Antikörper gegen das N-terminale Troponin I-Peptid
DE4243648 1992-12-23
PCT/EP1993/003661 WO1994015217A1 (fr) 1992-12-23 1993-12-22 Procede permettant de determiner des necroses du muscle cardiaque a l'aide d'anticorps diriges contre le peptide n-terminal de la troponine i

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WO1996010076A1 (fr) * 1994-09-28 1996-04-04 Spectral Diagnostics Inc. Anticorps monoclonal contre la troponine i cardiaque humaine
US6627404B1 (en) 1995-04-18 2003-09-30 Biosite, Inc. Methods for improving the recovery of troponin I and T in membranes, filters and vessels
US6991907B1 (en) 1995-04-18 2006-01-31 Biosite, Inc. Methods for the assay of troponin I and T and complexes of troponin I and T and selection of antibodies for use in immunoassays
FR2734267B1 (fr) * 1995-05-16 1997-08-01 Pasteur Sanofi Diagnostics Composition stabilisee de troponine pour immunoessais et procede de preparation d'une telle composition stabilisee
FR2756827B1 (fr) * 1996-12-05 1999-01-29 Pasteur Sanofi Diagnostics Composes synthetiques biepitopiques utilisables comme etalons dans les dosages biologiques de la troponine i
US6156521A (en) * 1997-12-19 2000-12-05 Biosite Diagnostics, Inc. Methods for the recovery and measurement of troponin complexes
US6248869B1 (en) 1997-05-29 2001-06-19 Medical Analysis Systems, Inc. Troponin I forms and use of the same
US7345142B2 (en) 2004-01-27 2008-03-18 Compugen Ltd. Nucleotide and amino acid sequences, and assays and methods of use thereof for diagnosis of cardiac disease
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